Arthur Hill

Squamous metaplasia amplifies pathologic epithelial-mesenchymal interactions in COPD patients

Squamous metaplasia (SM) is common in smokers and is associated with airway obstruction in chronic obstructive pulmonary disease (COPD). A major mechanism of airway obstruction in COPD is thickening of the small airway walls. We asked whether SM actively contributes to airway wall thickening through alteration of epithelial-mesenchymal interactions in COPD. Using immunohistochemical staining, airway morphometry, and fibroblast culture of lung samples from COPD patients; genome-wide analysis of an in vitro model of SM; and in vitro modeling of human airway epithelial-mesenchymal interactions, we provide evidence that SM, through the increased secretion of IL-1beta, induces a fibrotic response in adjacent airway fibroblasts. We identify a pivotal role for integrin-mediated TGF-beta activation in amplifying SM and driving IL-1beta-dependent profibrotic mesenchymal responses. Finally, we show that SM correlates with increased severity of COPD and that fibroblast expression of the integrin alpha(v)beta(8), which is the major mediator of airway fibroblast TGF-beta activation, correlated with disease severity and small airway wall thickening in COPD. Our findings have identified TGF-beta as a potential therapeutic target for COPD.

Mouse and human lung fibroblasts regulate dendritic cell trafficking, airway inflammation, and fibrosis through integrin αvβ8–mediated activation of TGF-β

The airway is a primary portal of entry for noxious environmental stimuli that can trigger airway remodeling, which contributes significantly to airway obstruction in chronic obstructive pulmonary disease (COPD) and chronic asthma. Important pathologic components of airway remodeling include fibrosis and abnormal innate and adaptive immune responses. The positioning of fibroblasts in interstitial spaces suggests that they could participate in both fibrosis and chemokine regulation of the trafficking of immune cells such as dendritic cells, which are crucial antigen-presenting cells. However, physiological evidence for this dual role for fibroblasts is lacking. Here, in two physiologically relevant models - conditional deletion in mouse fibroblasts of the TGF-β-activating integrin αvβ8 and neutralization of αvβ8 in human COPD fibroblasts - we have elucidated a mechanism whereby lung fibroblast chemokine secretion directs dendritic cell trafficking, in a manner that is critically dependent on αvβ8-mediated activation of TGF-β by fibroblasts. Our data therefore indicate that fibroblasts have a crucial role in regulating both fibrotic and immune responses in the lung.

Delayed primary repair of intrathoracic esophageal perforation: Is it safe?

The management of intrathoracic esophageal perforation with delayed diagnosis is a subject of controversy. Because of the obvious advantages of primary repair as a simple single-stage operation, this technique was preferentially used to treat 18 of 22 consecutive patients with esophageal perforation. These patients were stratified into three groups according to the time interval between perforation and repair: group A, less than 6 hours, five patients (28%); group B, 6 to 24 hours, six patients (33%); and group C, more than 24 hours, seven patients (39%). Group A patients were older (p < 0.05) and group B had fewer iatrogenic perforations (B, 17%; A, 80%; C, 57%, p < 0.1). Additional tissue was used to buttress the repair site in all three groups (A, 3/5 patients, 60%; B, 4/6 patients, 67%; C, 6/7 patients, 86%; p = not significant). In seven patients (39%), a fundic wrap was used to reinforce the site of primary repair. The outcomes of the three groups were analyzed. Group A had the lowest proportion of postoperative leaks (A, 0/4 patients, 0%; B, 4/6 patients, 67%; C, 5/6 patients, 83%; p < 0.05) and postoperative morbidity (A, 2/5 patients, 40%; B, 6/6 patients, 100%; C, 6/7 patients, 86%; p < 0.1). However the increased incidence of leak and morbidity did not lead to an increase in mortality. One death occurred in each group, with an overall mortality of 17% (A, 1/5 patients, 20%; B, 1/6 patients, 17%; C, 1/7 patients, 14%; p = not significant). We conclude that in the era of advanced intensive care capabilities, primary repair of intrathoracic esophageal perforation can be safely accomplished in most patients regardless of the time interval between perforation and operation. Leakage at the suture site is common unless primary repair is carried out without delay. Postoperative leakage, however, is usually inconsequential and does not necessarily result in an adverse outcome.

Integrin αvβ8-Mediated Activation of Transforming Growth Factor-β Inhibits Human Airway Epithelial Proliferation in Intact Bronchial Tissue

Transforming growth factor (TGF)-β is a potent multifunctional cytokine that is an essential regulator of epithelial proliferation. Because TGF-β is expressed almost entirely in a latent state in vivo, a major source of regulation of TGF-β function is its activation. A subset of integrins, αvβ8 and αvβ6, which are expressed in the human airway, has recently been shown to activate latent TGF-β in vitro, suggesting a regulatory role for integrins in TGF-β function in vivo. Here we have developed a novel, biologically relevant experimental model of human airway epithelium using intact human bronchial tissue. We have used this model to determine the function of integrin-mediated activation of TGF-β in the airway. In human bronchial fragments cultured in vitro, authentic epithelial-stromal interactions were maintained and integrin and TGF-β expression profiles correlated with profiles found in normal lung. In addition, in this model, we found that either the integrin αvβ8 or TGF-β could inhibit airway epithelial cell proliferation. Furthermore, we found that one mechanism of integrin-αvβ8-dependent inhibition of cell proliferation was through activation of TGF-β because anti-β8 antibody blocked the majority (76%) of active TGF-β released from bronchial fragments. These data provide compelling evidence for a functional role for integrin-mediated activation of TGF-β in control of human airway epithelial proliferation in vivo.

Selective Targeting of TGF-β Activation to Treat Fibroinflammatory Airway Disease

Therapeutic targeting of an extended-closed conformation of the integrin αvβ8 inhibits TGF-β activation and ameliorates symptoms of experimental airway disease in mice. Breathing Freely Narrowing of the airways through accumulation of scar tissue and inflammation results from chronic injury in common diseases such as chronic obstructive pulmonary disease (COPD) and severe chronic asthma. Such airway narrowing causes the obstruction responsible for the breathlessness that these patients experience, and there are no available treatments that ameliorate fibroinflammatory airway narrowing. In a new study, Minagawa et al. engineered a monoclonal antibody that locks in a specific inactive conformation of a protein named integrin αvβ8. This protein is a crucial receptor required for activation of transforming growth factor–β, a central mediator of pathological inflammation and fibrosis. This antibody, when administered to mice engineered to express only human and not mouse αvβ8, reduced airway inflammation and fibrosis in response to a variety of injurious agents including cigarette smoke and allergens that are involved in the pathogenesis of COPD. Airway remodeling, caused by inflammation and fibrosis, is a major component of chronic obstructive pulmonary disease (COPD) and currently has no effective treatment. Transforming growth factor–β (TGF-β) has been widely implicated in the pathogenesis of airway remodeling in COPD. TGF-β is expressed in a latent form that requires activation. The integrin αvβ8 (encoded by the itgb8 gene) is a receptor for latent TGF-β and is essential for its activation. Expression of integrin αvβ8 is increased in airway fibroblasts in COPD and thus is an attractive therapeutic target for the treatment of airway remodeling in COPD. We demonstrate that an engineered optimized antibody to human αvβ8 (B5) inhibited TGF-β activation in transgenic mice expressing only human and not mouse ITGB8. The B5 engineered antibody blocked fibroinflammatory responses induced by tobacco smoke, cytokines, and allergens by inhibiting TGF-β activation. To clarify the mechanism of action of B5, we used hydrodynamic, mutational, and electron microscopic methods to demonstrate that αvβ8 predominantly adopts a constitutively active, extended-closed headpiece conformation. Epitope mapping and functional characterization of B5 revealed an allosteric mechanism of action due to locking-in of a low-affinity αvβ8 conformation. Collectively, these data demonstrate a new model for integrin function and present a strategy to selectively target the TGF-β pathway to treat fibroinflammatory airway diseases.

Treatment of suture line bleeding with a novel synthetic surgical sealant in a canine iliac PTFE graft model

CoSeal mark surgical sealant (CoSeal) was evaluated for inhibiting suture line bleeding using a canine iliac PTFE graft model. Both iliac arteries of 12 heparinized canines were grafted with PTFE. CoSeal was applied to the suture lines of one graft in each animal. The contra-lateral graft served as a control and bleeding was controlled with gauze and pressure (tamponade). The cross-clamps were removed 30 s following application of CoSeal. Times to hemostasis and volume of blood loss at each graft site were determined. Compared to tamponade control, CoSeal significantly reduced the time to hemostasis (average of 5 min vs. greater than 15 min, p < 0.05) and blood loss (19 g vs. 284 g, p < 0.05). Small amounts of CoSeal were visible grossly or histologically at day 7. Histology showed moderate to marked inflammation in CoSeal sites and moderate inflammation in control sites at day 7. At 30 and 60 days, no CoSeal was visible grossly or histologically. Histology showed moderate inflammation in both CoSeal treated sites and in control sites at day 30 and mild to moderate inflammation in both CoSeal and control sites at day 60. CoSeal significantly reduced the time to hemostasis and blood loss in comparison to tamponade.

Interleukin-1β Induces Increased Transcriptional Activation of the Transforming Growth Factor-β-activating Integrin Subunit β8 through Altering Chromatin Architecture

Background: IL-1β acts on fibroblasts inducing TGF-β-dependent profibrogenic responses. Results: IL-1β increases expression of the TGF-β-activating integrin β8 subunit through altering nucleosomal positioning at the ITGB8 promoter. Conclusion: IL-1β increases accessibility of transcription factors to the ITGB8 promoter in lung fibroblasts through chromatin remodeling. Significance: This provides evidence for chromatin architectural changes mediating IL-1β profibrotic programs. The integrin αvβ8 is a cell surface receptor for the latent domain (LAP) of the multifunctional cytokine TGF-β. Through its association with LAP, TGF-β is maintained in a latent form that must be activated to function. Binding to the integrin αvβ8 with subsequent metalloproteolytic cleavage of LAP represents a major mechanism of TGF-β activation in vivo. Altered expression of the integrin β8 subunit (ITGB8) is found in human chronic obstructive pulmonary disease, cancers, and brain vascular malformations. We have previously shown that the proinflammatory cytokine interleukin-1β (IL-1β) increases ITGB8 expression on lung fibroblasts, which increases αvβ8-mediated TGF-β activation in fibrosis and pathologic inflammation. Here we report the mechanism of increased ITGB8 expression by IL-1β. Our data support a model where the chromatin architecture of the ITGB8 core promoter is altered by nucleosomal repositioning that enhances the interaction of an AP1 complex (containing c-Jun and ATF2). This repositioning is caused by the dissociation of HDAC2 with the ITGB8 core promoter, leading to increased histone H4 acetylation and a loosening of nucleosomal-DNA interactions allowing “opening” of the chromatin structure and increased association of c-Jun and ATF-2. These changes are mediated through NFκB- and p38-dependent pathways. Ultimately, these events culminate in increasing ITGB8 transcription, αvβ8 surface expression, and αvβ8-mediated TGFβ activation.

In vitro and in vivo studies of ePTFE vascular grafts treated with P15 peptide.

The purpose of this study is to evaluate the effectiveness of P15 cell-binding peptide treated ePTFE vascular grafts in vitro and in vivo. The P15 peptide was covalently immobilized onto ePTFE vascular grafts by an atmospheric plasma coating method. In vitro cell growth properties were studied using primary human umbilical vein endothelial cells (HUVECs) and primary human umbilical artery smooth muscle cells (HUASMCs). X-ray photoelectron spectroscopy and aminoacid analysis were used to analyze the surface characteristics of the peptide treated and untreated grafts. The cell growth study showed that the P15 peptide effectively promoted the adhesion and proliferation of endothelial cells. 700% more endothelial cells were proliferated on the P15-treated ePTFE grafts compared to the untreated ePTFE controls. In contrast, the P15 peptide was significantly less effective for promoting the adhesion and proliferation of smooth muscle cells than endothelial cells; only about 100% more smooth muscle cells proliferated on the P15-treated samples compared to the untreated control samples. The sheep model was used in the in vivo study. The amount of neointimal hyperplasia present at the arterial and venous sides of the anastomosis and the degree of endothelialization on the luminal surface of the grafts were assessed. Four P15-treated grafts and two control grafts were implanted as arteriovenous grafts between the femoral artery and vein or the carotid artery and jugular vein in two sheep (n = 6). The in vivo study showed that the thickness of the neointimal hyperplasia of untreated grafts was 3-times thicker than that of P15-treated grafts (P < 0.05) at the venous side of the anastomosis. P15-treated grafts also had a higher degree of endothelialization on the graft lumen.

Fetal lamb pulmonary hypoplasia: Pulmonary vascular and myocardial abnormalities

Neonatal pulmonary hypoplasia resulting from a congenital diaphragmatic hernia (CDH) produces hemodynamic changes and morphologic abnormalities of the pulmonary vasculature. To characterize the myocardial and pulmonary vascular status of the fetus with pulmonary hypoplasia, we studied four chronically instrumented, near-term fetal lambs with pulmonary hypoplasia, induced by producing a diaphragmatic hernia. We found an elevation in the pulmonary arterial pressure (control, 43.8 +/- 5.9 mmHg; CDH, 58.8 +/- 9.1 mmHg; p < 0.05), an elevation in the systemic arterial pressure (control, 43.8 +/- 0.48 mmHg; CDH, 58.6 +/- 6.7 mmHg; p < 0.05), and an elevation in the pulmonary vascular resistance (control, 0.47 +/- 0.11; CDH, 3.87 +/- 1.9; p < 0.05). In addition, though the total pulmonary blood flow was reduced (control, 83.5 +/- 32.9 mL/min; CDH, 22.2 +/- 17.6 mL/min; p < 0.05), the blood flow reduction was proportional to the reduction in the lung mass (control, 79.8 +/- 28.1 [in flow per 100-g lung weight]; CDH, 85.4 +/- 71.7). The increase in the pulmonary vascular resistance in relation to the unit lung mass (control, 0.55 +/- 0.33; CDH, 0.99 +/- 0.5) was not as pronounced as its increase in relation to the total pulmonary blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming Growth Factor-β and Interleukin-1β Signaling Pathways Converge on the Chemokine CCL20 Promoter

Background: Mechanisms that drive chronic inflammation in airway disease are not well understood. Results: We demonstrate a TGF-β-responsive enhancer element required for efficient IL-1β-dependent expression of the proinflammatory chemokine CCL20 by human lung fibroblasts. Conclusion: Convergence of TGF-β and IL-1β signaling pathways on the CCL20 promoter are required for efficient fibroblast expression of CCL20. Significance: These findings identify potential targets to reduce chronic inflammation in airway disease. CCL20 is the only chemokine ligand for the chemokine receptor CCR6, which is expressed by the critical antigen presenting cells, dendritic cells. Increased expression of CCL20 is likely involved in the increased recruitment of dendritic cells observed in fibroinflammatory diseases such as chronic obstructive pulmonary disease (COPD). CCL20 expression is increased by the proinflammatory cytokine IL-1β. We have determined that IL-1β-dependent CCL20 expression is also dependent on the multifunctional cytokine TGF-β. TGF-β is expressed in a latent form that must be activated to function, and activation is achieved through binding to the integrin αvβ8 (itgb8). Here we confirm correlative increases in αvβ8 and IL-1β with CCL20 protein in lung parenchymal lysates of a large cohort of COPD patients. How IL-1β- and αvβ8-mediated TGF-β activation conspire to increase fibroblast CCL20 expression remains unknown, because these pathways have not been shown to directly interact. We evaluate the 5′-flanking region of CCL20 to determine that IL-1β-driven CCL20 expression is dependent on αvβ8-mediated activation of TGF-β. We identify a TGF-β-responsive element (i.e. SMAD) located on an upstream enhancer of the human CCL20 promoter required for efficient IL-1β-dependent CCL20 expression. By chromatin immunoprecipitation, this upstream enhancer complexes with the p50 subunit of NF-κB on a NF-κB-binding element close to the transcriptional start site of CCL20. These interactions are confirmed by electromobility shift assays in nuclear extracts from human lung fibroblasts. These data define a mechanism by which αvβ8-dependent activation of TGF-β regulates IL-1β-dependent CCL20 expression in COPD.